Gene transfer to pulmonary epithelia is a direct approach to correct the defects associated with cystic fibrosis (CF). Problems currently limiting the clinical application of this method include poor apical gene transfer efficiency, transient expression, low vector titers, adverse immune responses, and a limited understanding of the intracellular fate of integrating vectors. Our work focuses on the development and application of recombinant lentiviral vectors based on feline immunodeficiency virus (FIV) to surmount these problems. For many viral vectors, the cellular receptors are functionally absent from the apical surface of airway epithelia. The post entry interactions between lentiviruses and airway epithelia, including integration, have received little study. We recently successfully pseudotyped FIV using the glycoproteins (GPs) from the filoviruses (Marburg/Ebola), and modified the GPs to improve vector titer. Importantly, FIV vector pseudotyped with filovirus GPs preferentially enters polarized human airway epithelia from the apical surface without the disruption of tight junctions. Therefore, we have an important reagent to address key issues regarding vector-host cells interactions. Our central hypothesis is that pseudotyped FIV-based lentiviral vectors can enter airway epithelia via the apical surface in vitro and in vivo and achieve gene transfer that persists and corrects the CF defect. There are three specific aims: Aim 1. Does targeting apical entry with FIV containing a filovirus glycoprotein envelope confer persistent transgene expression in human airway epithelia? We will investigate vector-host cell interactions and address questions relevant to their application using models of well-differentiated human airway epithelia. These include: What percent of cells and what cell types are transduced? Will gene expression persist following apical gene transfer? How do vector copy number and expression correlate over time? Aim 2. Can FIV pseudotyped with filovirus glycoproteins lead to lasting correction of the ion transport defects in CF airway epithelia? We will use an optimized apical targeting FIV construct expressing CFTR to transduce well-differentiated human CF epithelia. Important questions to address include: Does correction persist? Does integration correlate with persistence of correction of the ion transport defects? Is vector copy number/cell stable in cells demonstrating long-term correction? Aim 2. What is the fate of the integrated FIV provirus in airway epithelia? Stable transgene expression can be achieved in vitro and in vivo with lentiviral vectors, yet nothing is known regarding the fate of the FIV proviral DNA in human airway epithelia. To begin to address the specificity of integration for the FIV vector system, we will use PCR and microarray hybridization to sequence and map FIV integration sites in the human genome and extend these experiments to the mouse model. Completing these three aims will address key questions regarding host-vector interactions and improve our ability to apply lentiviral vectors for diseases involving airway epithelia.